The number of patients suffering blindness due to a retinal degenerative disease such as age-related macular degeneration or retinitis pigmentosa has been increasing. Because a disorder of photoreceptors is the direct cause of blindness in these diseases, production of photoreceptors (or photoreceptor precursors) in vitro would possibly contribute significantly to research into these diseases and development of therapies for these diseases.
Including the present inventors, many groups have attempted to generate photoreceptors from iris tissue (non-patent documents 1, 2), ciliary tissue (non-patent document 3), or embryonic stem (ES) cells (non-patent documents 4-6). Compared with tissue stem cells, ES cells have the capability of proliferating infinitely and permit production of sufficient numbers of cells for research and treatment, and are therefore superior. Recent studies have demonstrated that retinal progenitor cells can be efficiently produced from ES cells in vitro. By contrast, the in vitro generation of photoreceptors from ES cell-derived progenitor cells remains inefficient unless the progenitor cells are co-cultured with developing retinal tissue. For example, in a previous report by the present inventors (non-patent document 6), the present inventors showed efficient induction (up to 16%) of neural retinal progenitor cells from mouse ES cells using a serum-free floating culture of embryoid body-like aggregates (SFEB) system combined with treatments with Dkk1, LeftyA, serum and Activin (SFEB/DLFA) (non-patent document 7). ES-derived neural retinal progenitor cells, when co-cultured with embryonic retinal tissue, are capable of producing photoreceptors. However, no method has been established for producing photoreceptors under culture conditions without the co-culture. In particular, a defined method of culture wherein photoreceptors are efficiently produced from human ES cell is expected to overcome the definitive limitation on transplantation therapy; there is demand for the development of the method.
In the document 7, the present inventors studied for effects of exogenous factors (Fgf, taurine, shh, and /RA) on SFEB/DLFA-treated cells without sorting, and were unable to find an evidently positive effect on the differentiation into rhodopsin+ photoreceptors. For induction of retinal progenitor cells from mouse embryonic stem cells, addition of FCS was essential.    non-patent document 1: Haruta, M. et al. Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue. Nat. Neurosci. 4, 1163-1164 (2001).    non-patent document 2: Sun, G. et al. Retinal stem/progenitor properties of iris pigment epithelial cells. Dev. Biol. 289, 243-252 (2006).    non-patent document 3: Tropepe, V. et al. Retinal stem cells in the adult mammalian eye. Science 287, 2032-2036 (2000).    non-patent document 4: Zhao, X., Liu, J. & Ahmad, I.Differentiation of embryonic stem cells into retinal neurons. Biochem. Biophys. Res. Commun. 297, 177-184 (2002).    non-patent document 5: Hirano, M. et al. Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells. Dev. Dyn. 228, 664-671 (2003).    non-patent document 6: Ikeda, H. et al. Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells. Proc. Natl. Acad. Sci. USA 102, 11331-11336 (2005).    non-patent document 7: Watanabe, K. et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288-296 (2005).